Nanosilica modulates C:N:P stoichiometry attenuating phosphorus toxicity more than deficiency in Megathyrsus maximus cultivated in an Oxisol and Entisol

Silicon (Si) nanoparticles can attenuate nutritional disorders caused by phosphorus in forages through nutritional homeostasis. This paper aims to evaluate the effects of P deficiency and toxicity in Megathyrsus maximus cultivated in two types of soils and to verify whether Si application via fertigation can mitigate these imbalances. The following two experiments were carried out: cultivation of forage plants in pots with Entisol and Oxisol, in a 3 × 2 factorial design, with three nutritional levels of phosphorus (deficient, adequate, and excessive) and two Si concentrations in the irrigation water (0 and 1.5 mmol L−1). Height, number of tillers, rate of leaf senescence, dry matter production, C:N, C:Si, C:P, and N:P ratios; and C, P, and N use efficiencies were evaluated in two growth cycles. P imbalances hampered carbon assimilation, C:N:P homeostasis, and dry matter production. Nanosilica fertigation promoted silicon uptake, improving C:N:P homeostasis and nutritional efficiency in plants under P deficiency and toxicity. Leaf senescence was reduced with addition of Si in plants grown in Oxisol in the three nutritional states of P. Silicon attenuated the stress caused by P toxicity in Entisol and Oxisol, improving production in plants without nutritional stress in Oxisol. The supply of Si nanoparticles in the cultivation of M. maximus can contribute to a more efficient and sustainable use of phosphorus in pastures.

Fertilization was carried out through soil application by supplying K, S, Zn, and B at doses of 150, 50, 5, and 0.5 mg dm −3 , respectively, besides supplying 5 mg dm −3 of Fe to the Entisol, using the following sources: potassium chloride, calcium sulfate, zinc sulfate, boric acid, and iron chelate. Phosphorus was applied at doses of 0, 200 53 , and 600 mg dm −3 in the plots corresponding to the deficient, adequate, and excessive levels of P, respectively, in the form of triple superphosphate. Nitrogen fertilization was carried out by N topdressing with 300 mg dm −3 of N in installments during each regrowth period, being always incorporated with irrigation.
Sowing was carried out directly in the soil of each plot by manually depositing approximately 20 seeds of M. maximus cv. Zuri in a circular groove at a depth of 1 cm. After seedling emergence, thinning was performed, maintaining two plants per pot. At 30 days after sowing, a uniform cut was carried out at 17 cm from the ground level in all pots to stimulate tillering, initiating the splitting of nitrogen fertilization and Si application, as well as the period of data collection. On that occasion, 4 mg dm −3 of P were applied to the plants in the deficient treatment (Entisol) to enable the minimum growth of these plants.
Experimental conditions. The temperature and relative humidity inside the greenhouse were monitored daily using a thermo-hygrometer. The maximum average temperature recorded was equal to 46.3 ± 5 °C, with a minimum average temperature of 21.8 ± 3 °C and average relative humidity equal to 47.6 ± 10%.
The water-holding capacity or available water capacity (AWC) of the soils was determined. Two pots filled with each soil were subjected to saturation in a container with water for 24 h. After saturation, the pots were covered with plastic and removed from the saturated environment for free drainage. After draining, soil samples were dried in an oven and the AWC was determined by the difference between the masses of drained (wet) and dry soil. The management of water replacement was established by maintaining the soils at 70% of AWC, a condition indicated for Poaceae 54 , under which water is available and gas exchanges are maintained in the root zone. The pots were weighed daily in the late afternoon, after which evapotranspirated water was replaced 55 .
Silicon supply. Silicon was supplied daily from the moment of sowing until the last cut of the plants, using a solution of 1.5 mmol L −1 of Si 36 . The solution was prepared with colloidal nanosilica (particle size between 8.5 and 9.7 nm, specific surface area of 300 m 2 g −1 , and pH 10.5). The same volume of solution was applied in all treatments, with the volume being defined based on the treatment with the smallest daily water demand. Thus, in treatments with greater water demand, irrigation was supplemented with deionized water. The volume of solution applied was quantified at the end of the experimental period, totaling 26.2 and 22.3 L of solution applied per pot, providing 1.09 and 0.94 g per pot of Si in the Oxisol and Entisol, respectively. In plots that did not receive Si, irrigation was always performed with distilled and deionized water.
Analysis. Height, tillering, and senescence of plants. At the end of each of the two regrowth cycles, plant height (cm) was measured considering the length from the base to the inflection of the fully developed leaves, using a ruler. The number of tillers, as well as green and senescent leaves were counted to calculate the percentage of senescent leaves per pot (%).
Biomass production. The plants were cut twice when reaching 70 cm in height in the treatment with adequate P content 56 . This height was reached at 25 and 31 days after the uniform cut in the Entisol and Oxisol, respectively, when the first cut was made. At 28 and 32 days of regrowth in the Entisol and Oxisol, respectively, the second cut was performed. The cut was performed using scissors, leaving 30 cm of residual material above the ground and collecting only the mass of the grazing strata of plants. Then, samples were washed in detergent solution (0.1% v:v) and deionized water, successively, being dried in an oven with forced air circulation (65 ± 5 ºC) until reaching constant mass. After reaching constant mass, shoot dry matter per plot was determined (DM, g per pot). The dried material was then passed through a Willey mill.
Contents of C, N, P, and Si. C content was determined through wet digestion of the dry matter with a K 2 Cr 2 O 7 solution and titration with FeSO 4 by the modified Walkley-Black method 57 . To evaluate N content, the sample was submitted to digestion with sulfuric acid, distillation by the Kjeldahl method, and determination by titration 58 . P was determined from nitric-perchloric acid digestion and reading in a spectrophotometer at 420 nm 59 . Si was extracted with alkaline digestion in hydrogen peroxide at 120 °C 60 and determined by reading in a spectrophotometer at 410 nm after a colorimetric reaction with ammonium molybdate 51 . The contents of each element were expressed in g kg −1 .
Stoichiometric ratios and use efficiency. C:N, C:P, N:P, and C:Si stoichiometric ratios were calculated using the contents of the elements in the dry matter. The use efficiencies of C, N, and P were calculated using the expression: ((total dry matter produced) 2 /(total accumulation of nutrient in the plant)) 61 .

Results
P, Si, C, and N contents. In most situations evaluated (cuts and soils) there was a significant P x Si interaction (p < 0.05) in the P, Si, C and N contents, that is, Si influences these contents differently depending on the state of P. Similarly, the effect of P state can also be influenced by the absence or presence of Si. Plants cultivated in the condition of P deficiency and excess P in the absence of Si showed lower and higher levels of the element in the plant, respectively, in both soils and forage cuts (Fig. 1a-d). In the absence of Si, the nutritional status of P influenced the Si content in the plants, resulting in a higher Si content in plants under P deficiency and excess P in Entisol in the first cut and under excess P in the second cut, as well as in plants under P deficiency of P in Oxisol in the first cut ( Fig. 1e-h). The C content in plants grown without Si application was lower under P deficiency in both cuts and under excess P in the second cut in Oxisol (Fig. 1i,k). In the cultivation in Entisol in the absence of Si, a lower C content was observed in treatments with excess P in both cuts, as well as under deficiency in the first cut (Fig. 1j,l). In the absence of Si, there was higher N content in the plant under excess P in both cuts and soils ( Fig. 1m-p).
In the presence of Si in Oxisol, a reduction in P content was observed in the condition of excess P in the second cut (Fig. 1c), while in plants cultivated in Entisol there was a higher P content in P-deficient plants and a lower P content in P-excess plants, as well as a lower P content in plants with excess P in both cuts (Fig. 1b,d). The application of Si in relation to its absence promoted a higher Si content in the plant for all nutritional levels of P, soils, and forage cuts ( Fig. 1e-h). The C content of plants was lower in the presence of Si for all nutritional levels of P in the first cut in Oxisol and in the second cut in Entisol, as well as under excess P in the first cut in Entisol and in the deficiency and sufficiency of P in the second cut in Oxisol ( Fig. 1i-l). In the presence of Si, there was a lower N content in plants under P sufficiency in both cuts and under excess P in the second cut in Oxisol (Fig. 1m,o), as well as in plants under excess P grown in Entisol in both cuts (Fig. 1n,p). C:Si, C:N, C:P and N:P stoichiometric ratios. The P x Si interaction was significant (p < 0.05) for the C:Si ratio in both soils and cuts, and in at least one cut in each soil type for the C:N, C:P and N:P ratios. Cultivation with the absence of Si resulted in the lowest value of the C:N ratio in plants under P deficiency and excess P in both soils, with the exception of the deficiency in the second cut in Entisol, in which the C:N ratio was not affected (Fig. 2a-d). The absence of Si in forage plant cultivation also resulted in higher C:N and C:Si ratios in the adequate nutritional status of P in both soils, decreasing in plants with P imbalances, except for the second cut of plants under excess P in Oxisol and under P deficiency in Entisol ( Fig. 2a-h). The highest C:P and N:P ratios occurred in plants under deficiency, while the lowest value of these ratios occurred in plants with excess P, respectively, in both cuts and soils ( Fig. 2i-l; m-p).
Fertigation with Si provided a lower C:Si ratio for plants, regardless of soil type or cut ( Fig. 2e-h). In plants grown in Entisol, the presence of Si resulted in the lowest C:P and N:P ratios under P deficiency in both cuts, while resulting in the highest value of these stoichiometric ratios in plants grown under excess P in the second cut ( Fig. 2j,l,n,p). In plants cultivated in Oxisol, the presence of Si resulted in an increase in the N:P ratio under the adequate level of P in the second cut of the grass forage ( Fig. 2i,k,m,o).
Use efficiency of P, C, and N. The P x Si interaction was significant (p < 0.05) for P use efficiency in the second cut in both soils. In the absence of Si fertigation, P use efficiency by the plants was higher under nutrient deficiency in both cuts and soils, except for the first cut in Oxisol ( Fig. 3a-d). P deficiency and excess P in the forage grass resulted in lower C and N use efficiency by the plants in both soils and cuts, with the exception of P deficiency in Entisol in the second cut, in which N use efficiency was not affected (Fig. 3e-l).
When there was the addition of Si, a greater value for P use efficiency was observed in plants under the three nutritional levels of P in the first cut, as well as in plants under P deficiency in the second cut and cultivated in Entisol (Fig. 3b,d). In plants grown in Oxisol, the presence of Si resulted in a higher P use efficiency only under the adequate and excessive levels of P in both cuts (Fig. 3a,c), while higher C use efficiency was observed in plants under P deficiency in the second cut ( Fig. 3g) and in the condition of excess P in both forage cuts (Fig. 3e,g). In plants grown in Entisol, fertigation with Si resulted in the highest C use efficiency in plants under both P imbalances, being restricted to the second cut (Fig. 3h). There was an increase in N use efficiency in plants grown with Si in adequate nutritional status and excess P grown in Oxisol (Fig. 3i,k), as well as in plants with excess P grown in Entisol in both cuts (Fig. 3j,l). There was no effect of Si on N use efficiency of P-deficient plants in any of the evaluated soils and cuts ( Fig. 3i-l).
Height, tillering, senescence, and dry matter production. For the parameters plant height and percentage of senescent leaves, there was a significant P x Si interaction (p < 0.05) in the second cut in Oxisol, and also for the number of tillers in Entisol, while for the dry matter production the factors acted independently. In the absence of Si, plant height presented a lower value in the nutritional condition of excess P in both soils and cuts, as well as in Entisol under P deficiency in both cuts in relation to plants with adequate P status ( Fig. 4a-d).
In the absence of Si, the lowest value for the number of tillers occurred in plants under P deficiency in both soils and cuts ( Fig. 4e-h), while the highest value occurred in plants under excess P grown in Oxisol in the first cut (Fig. 4e). Excess P resulted in the highest rate of foliar senescence, except for the first cut in Oxisol ( Fig. 4i- www.nature.com/scientificreports/ Shoot dry matter production in the absence of Si showed the lowest value in P-deficient plants grown in Oxisol in both cuts (Fig. 4m,o), as well as in plants grown in Entisol in the first cut (Fig. 4n). In plants under excess P and under the absence of Si, the lowest dry matter production occurred in Entisol and in both cuts (Fig. 4n,p). However, when plants were cultivated in Oxisol, this occurred only in the first cut (Fig. 4m).
Under excess P, plants fertigated with Si showed an increase in height in both soils and cuts, except for the first cut in Oxisol (Fig. 4a-d). In plants grown in Oxisol, there was a greater number of tillers in the adequate level of P in both cuts (Fig. 4e,g), as well as under excess P in the first cut in relation to the absence of Si (Fig. 4e). In plants grown in Entisol in the presence of Si, there was a greater number of tillers in plants under excess P in both cuts (Fig. 4f,h). www.nature.com/scientificreports/ Under excess P, plants fertigated with Si showed an increase in height in both soils and cuts, except for the first cut in Oxisol (Fig. 4a-d). In plants grown in Oxisol, there was a higher number of tillers under the adequate level of P in both cuts (Fig. 4e,g), as well as under excess P in the first cut in relation to the absence of Si (Fig. 4e). In plants cultivated in Entisol and in the presence of Si, there was a greater number of tillers in plants under excess P in both cuts (Fig. 4f,h).
The rate of leaf senescence decreased with the addition of Si in relation to its absence in plants grown in Oxisol in the three nutritional levels of P and both cuts (Fig. 4i,k). In plants cultivated in Entisol, the rate of leaf senescence decreased with the addition of Si in plants under excess P in both cuts, as well as in the adequate level of P in the second forage cut (Fig. 4j,l). Si supply increased the dry matter production of plants under excess P www.nature.com/scientificreports/ grown in Entisol in both cuts (Fig. 4n,p), as well as in plants grown under adequate and excessive levels of P in Oxissol, in both cuts (Fig. 4m,o), with no effect on macronutrient deficiency in any of the soils or cuts.

Discussion
Biological damage caused by P imbalances in the plant without Si supply. The deficiency of P in the forage was clear, as plants cultivated without P application showed low P levels both in the plant cultivated in Oxisol (3.4 g kg −1 ) and in Entisol (7.8 g kg −1 ) in relation to the plant grown under the adequate P level in Oxisol (7.2 g kg −1) and Entisol (19.1 g kg −1 ). P deficiency in the plant was expected, as the soils used in this experiment presented low levels of available P. This is common in weathered soils, especially in Oxisol, which has a high phosphate adsorption capacity 66,67 . Studies on the deficiency of P in forage plants are restricted to the evaluation of nutrient levels and symptoms [68][69][70] , not analyzing whether this disorder causes stoichiometric changes. Thus, for the first time in a forage plant, it was verified that P deficiency decreased the C:Si ratio in plants, especially in the first cut, in plants grown in sandy or clayey soil. This is because plants deficient in P presented a higher Si content compared to those under sufficiency due to greater Si absorption even without application of the element. This result confirms that the forage plant develops the ability to absorb natural Si from the soil when subjected to nutritional stress 24 . It is possible that the increase in Si absorption may have been favored by the liming of the soil, as phytoliths (a material rich in Si previously deposited in the soil) can be dissolved through an increase in the pH of the solution 71 , favoring the availability of the element in the soil. However, in the second cycle there was a decrease www.nature.com/scientificreports/ in Si content in these P-deficient plants compared to the first forage cycle, confirming that although phytoliths are a source of Si 72,73 , they do not sustain Si uptake over successive cuts. P deficiency by decreasing the C content of the plants compared to those under sufficient or adequate levels of P reflected in a decrease in the C:N ratio of the forage in both soils and cuts, except for the second cut in plants grown in Entisol. The decrease in C content in P-deficient plants observed in this study is similar to that reported for another species (Holcus lanatus) 74 , which confirms the essential role played by P in the assimilation of C, as it regulates photosynthesis by acting as substrate, product, and/or effector of enzymes of the C4 Figure 4. Plant height, number of tillers, rate of leaf senescence, and biomass production in the grazing strata, in the first (a,b,e,f,i,j,m,n) and second cuts (c,d,g,h,k,l,o,p), in the forage plant M. maximus cv. BRS Zuri cultivated under phosphorus levels of deficiency (− P), adequacy (P), and excess (+ P) in Oxisol and Entisol, in the absence (− Si) and presence (+ Si, 1.5 mmol L −1 ) of silicon. Ns, * and ** correspond to the non-significant F-test, significant at 5 and 1%, respectively. Means were tested by the SNK test at 5% error probability. Uppercase letters compare means between nutritional levels and lowercase letters compare the effect of Si. Bars represent the standard error of the mean, n = 4. www.nature.com/scientificreports/ pathway in mesophyll cells 68,75 . Thus, even with a possible recycling of P in deficient plants 8 , it is not enough to meet the demand of the plant organs generating nutrient deficiency, and its damage is well documented in photosynthetic efficiency 47 . Changes in C:N:P stoichiometric ratios in deficient plants resulted in greater P use efficiency compared to plants grown under P sufficiency. This is natural in plants grown in soils with restricted P contents due to mechanisms such as membrane remodeling with P-free lipids (such as galacto-and sulfolipids) 70 or due to the preservation of P in inorganic forms, facilitating its remobilization 76 . However, there was a decrease in the use efficiencies of N and C because the conversion of N and C into biomass depends on P cellular homeostasis, as it is essential to the physiological and biochemical processes in plant cells, being a component of nucleic acids and membrane lipids, as well as a mediator of the energy metabolism 77,78 .
The efficiency of plants to use absorbed nutrients to compose its tissues has a direct influence on biomass production 61,79 . Therefore, P deficiency in relation to P-sufficient plants reduced nutritional homeostasis and the use efficiency of C and N, affecting forage plant growth in both cycles and in the two soils studied, with a decrease in dry matter production dries of 50 and 31% in plants grown in Oxisol and Entisol, respectively. These losses have been commonly reported in Poaceae cultivated under low levels of P 44,80 , limiting the productive capacity of pastures in tropical soils 3,4 .
Another important nutritional disorder in plants is P toxicity. It is still little reported in forage grasses, especially in crops without silicate fertilization, which is the condition of most pastures in the world. In this research, it was clearly evident that the high dose of P applied reflected in high average levels of P in the plant, reaching approximately 18.3 and 44.2 g kg −1 compared to the treatment under P sufficiency, which presented 7.2 and 19.1 g kg −1 of P in plants grown in Oxisol and Entisol, respectively. It should be noted that the lower P content in plants grown in Oxisol compared to Entisol is probably due to the greater adsorption of the element in Oxisol, reducing P availability in the soil and its absorption by the plant 80 . The opposite occurred in Entisol, which provided high levels of P in the plant due to the low clay content in this soil, giving it a low adsorption capacity for phosphate and greater availability for the plant, with a greater risk of toxicity in the plants 81,82 .
It is worth mentioning that the stress resulting from excess P in the plant stimulated the absorption of residual Si from the soil by the forage plant (as it occurred under P deficiency). This confirms that the plant uses the strategy of absorbing Si even at low levels in the soil, which may be a plant defense mechanism to mitigate stresses 24,83 .
An aspect not yet reported in forage plants is the effect of toxicity or excess P on C:N:P stoichiometry. It was evidenced in this research that the excess P in relation to the sufficiency of P affected the C:N:P stoichiometric ratios in the forage, with a decrease in the C:P, N:P , and C:N ratios in the two studied soils. The C:N ratio is highlighted for decreasing on average 28 and 49% in plants grown in Oxisol and Entisol, respectively. This reduction was due to the combination of a lower C content and a higher N content in plants under excess P, inhibiting the activation of RubBisCO by decreasing photosynthesis 15,84 .
In addition, we also showed that lower C:P and N:P ratios may indicate that C and N can limit P toxicity in the plant. Although there was a greater N uptake, the generated stoichiometric imbalance limits the use of nutrients 85 . Thus, the loss of C:N:P homeostasis had nutritional consequences on the plant, as it decreased the use efficiencies of P, C, and N in plants grown in both soils and cuts because the conversion of nutrients into biomass depends on the possibility and/or capacity of the plant to maintain sufficient concentrations while maintaining in stoichiometric equilibrium in the tissues 19,86 .
As a result of the decrease in nutrient use efficiencies, excess P reduced plant growth, decreasing plant height and increasing the rate of leaf senescence in plants cultivated in both soils, thus causing a 24% decrease in dry matter production in the two soils and two cuts of forage cultivated in Entisol, while causing a 23% decrease in the first cycle of plants cultivated in Oxisol. Thus, the physiological damage of P toxicity (especially in photosynthesis) 17,84 was previously induced by nutritional disturbances in elemental homeostasis.
Reflections of P imbalance were observed for the first time in the species M. maximus. P toxicity begins with an excess of thin tillers and leaves with reduced length, not very rigid and decumbent (Fig. 5a,b). In addition, there was chlorosis in older leaves, with purplish spots that progress to necrosis from the apex to the base in the leaf blade (Fig. 5c), bein similar to what was described in leaves of wheat and rice plants under P toxicity 84,87 .
Thus, the observed results allow accepting the first and second hypotheses tested, as it was shown that P imbalances modify the C:N:P stoichiometry of the forage and that P toxicity caused a greater stoichiometric imbalance especially in plants cultivated in a sandy soil (Entisol). Therefore, the risk of facing P toxicity is high for forages grown in Entisol, requiring a rigorous management of this nutrient.

Benefits of Si in mitigating stress under P deficiency and toxicity in grass forage.
Si is a wellknown element to attenuate different abiotic stresses in many crops. However, in forage plants, studies evaluating P deficiency were carried out only in hydroponic cultivation 36,44 , and there is a lack of reports of crops in different soils, especially regarding P toxicity. This is worrying, as forage crops are grown in different soils worldwide, with vast areas cultivated in Oxisol and Entisol. Thus, it is important to know if Si is really capable of mitigating these stresses under these cultivation conditions.
Initially, it was evident that plants cultivated under P deficiency in Entisol and that received the application of Si nanoparticles presented an increase of 21% in the P content in relation to those that did not receive Si. It is important to note that the levels of Si in the plant were higher in deficient plants than in plants under P sufficiency as a in response to stress. Therefore, a higher Si content in the soil, and consequently in the plant, has the ability to positively regulate the gene expression of P transporters, in addition to favoring the availability of P in the soil by stimulating the exudation of malate and citrate in Poaceae 88 , decreasing the adsorption and favoring the uptake of P by the plant. In this context, the use of Si can favor a greater use of the P present in the www.nature.com/scientificreports/ soil by pastures, which is essential, since nutrient reserves are finite 89 25% of the land cultivated with pastures has soils with low P content 1,4 . Additionally, Si promoted a decrease in C:P and N:P ratios in the two cycles in Entisol, thus promoting a 9% increase in the P use efficiency by the plant. This was also reflected in greater C use efficiency in the second forage cut in both soils. Complementarily, we carried out a Pearson correlation analysis between the studied variables and the DM production of the deficient plants, and we observed that the use efficiencies of C (r = 0.996 * ) and P (r = 0.991 * ), as well as the C:P ratio (r = 0.986 * ) were the ones that most contributed to DM production in both soils. A similar action was observed in rice plants deficient in N, in which Si promoted greater nutritional homeostasis, contributing to the efficient use of nutrients 35 . The benefits of Si in the balance of C:N:P stoichiometry were not enough to increase the efficiency of N use in the forage in the two cuts and in the two soils. Consequently, there was no increase in the number of tillers or in dry matter production by plants deficient in P. However, other authors have observed this effect of Si in other pastures 46 and in sorghum 47 . It is possible that the effect of Si on C:N:P homeostasis did not reach a sufficient level to favor greater accumulation of biomass and mitigate P deficiency in the forage within the growth cycle studied. Considering the potential of Si presented in our study, we reinforce the need for further research aimed at studying the mitigating action of Si on P-deficiency stress in Poaceae plants.
It was verified that P toxicity was severe in plants that did not receive Si, showing a limiting nutritional disorder. In this research, we observed that in plants cultivated under excess P, the increase in the absorption of the Si applied to the soil in the form of nanosilica reduced the P content in the two cuts of the forage cultivated in Entisol, as well as in the second cut in Oxisol, revealing that Si negatively modulates P absorption in conditions www.nature.com/scientificreports/ of excess. One of the possible ways to attenuate P toxicity by Si is the modulation of the gene expression of P transporters 90 , which has already been demonstrated in rice 8 and depends on the translocation and accumulation of Si in the shoot 91 . Forages have a good capacity for Si absorption, as they are a Si-accumulating plants 29 , especially when the element is supplied in the form of nanosílica 92 . The increase in Si absorption in relation to its absence in P-deficient plants caused changes in stoichiometric ratios in the forage, especially in the second cut of the plant cultivated in Entisol, with lower C:P and N:P ratios, which is due to the greater P uptake. Furthermore, Si increased the C:N ratio in the forage in both cuts in Entisol, improving stoichiometric homeostasis. Therefore, for the first time there is a report on the potential of Si to modify C:N:P stoichiometry in forage plants cultivated under excess P, but this depends on the soil used for cultivated, as it stood out more in Entisol, probably due to the greater severity of macronutrient toxicity. Research on the effects of Si on C:N:P stoichiometry in stressed plants 93 , of water deficit in sugarcane 43,55,94 , and of the effect of Si in the forage plant M. maximus are recent 38 .
The effects of Si in relation to its absence on the C:N:P homeostasis of forage plants cultivated under P toxicity was sufficient to increase nutritional efficiency, highlighting the efficiency of N use, providing an average increase of 18% in this efficiency, in the two cycles and soils. There was also an increase in the use efficiency of C and P depending on the forage cut. These added effects on nutritional efficiency were sufficient to favor forage growth under excess P by increasing height and number of tillers in both soils, consequently increasing forage dry matter production. In Pearson's correlation analysis, we observed that the use efficiencies of C (r = 0.996 * ), P (r = 0.979 * ) and N (r = 0.945 * ) in Entisol, and the efficiencies use of C (0.996 * ), and N (r = 0.949 * ) and plant height (r = 0.901 * ) in Oxisol, were the ones that most contributed to the increase in DM in plants under P toxicity.
An important aspect favored by Si to attenuate P toxicity and plant growth was its strong and constant effect of decreasing the rate of leaf senescence in all soils and growth cycles. It is possible that this effect of Si on leaf senescence may be related to the increase in N use efficiency, as this nutrient increases the period when leaves are photosynthetically active 78 and also deletes genes related to mechanisms for the induction of senescence 95 . This effect of Si in reducing leaf senescence is little studied, being only reported in plants cultivated under water deficit, such as rice 96 and sorghum 97 , and under sulfur deficiency, such as in barley plants 95 . In this paper, this effect was visually noticed (Fig. 5a,b,c).
It was also possible to visually notice the benefits of Si in mitigating P toxicity, since the damage caused by this previously described nutritional disorder was not observed when the plants received Si (Fig. 5), which presented better plant architecture, as already observed in rice plants 98 due to greater rigidity in the plant tissues 73 .
In parallel, another benefit of Si in the plant would be its effect of increasing the energy in plant metabolism, which would favor the antioxidant defense mechanisms of plants due to P toxicity, providing components for the Poaceae cell wall, ensuring structural resistance, and reducing the need for lignin synthesis (which demands a high energy content) 99,100 . This energy can be used in the organic synthesis of plant defense compounds against stress.
Our results indicate that forages are sensitive to P toxicity, causing an imbalance of C:N:P stoichiometry in the plant, and that the use of nanosilica via fertigation can mitigate this stress, making the pasture productive again and providing environmental implications. Excess P in the soil is at risk of contaminating water systems [101][102][103] , and with forage cultivation under fertigation with Si, the P extracted from the soil would be continuously exported in the forage, without risk to the environment.
Thus, the third hypothesis of this research can be partially accepted, since fertigation with Si enabled to attenuate the excess P in both soils, but its benefits were not sufficient to mitigate P deficiency in the evaluated period. This reinforces in an unprecedented way that Si is more effective to attenuate P toxicity in relation to P deficiency in this species.
Benefits of Si in forage plants under P sufficiency. Surprisingly, it was observed that in plants cultivated under P sufficiency, the supply of Si resulted in a change in stoichiometry, balancing the C:N and N:P ratios, especially in forages cultivated in Oxisol, also balancing the C:P ratio in Entisol in the second cut. Therefore, the potential of Si to increase nutritional efficiency in forages is unveiled due to greater stoichiometric homeostasis, especially of C:N and N:P. These changes in stoichiometry resulted in an increase in the use efficiency of P and N in the two forage cuts with sufficient P, associated with a decrease in the rate of senescent leaves and an increase in tillering. Consequently, there was an increase of approximately 5% in the dry matter production of plants cultivated in Oxisol. In Entisol, stoichiometric changes were restricted and were not sufficient to increase dry matter production in plants under sufficiency of P.
The role played by silicon as a facilitator of the balance of mineral nutrients in the plant and a mediator of morphological and biochemical changes 104,105 may explain the increase in plant growth even without nutritional stress. Furthermore, high levels of Si in the shoots, such as those found in this study, favor the expression of the benefits of Si to plants, with recent studies reporting production gains due to stoichiometric balance and efficiency in the use of macronutrients such as N and P in different species cultivated without stress, such as forage plants cultivated in a hydroponic system 36,44 , sugarcane 37,106 , and sorghum 107 .
These results have an important practical implication, as they allow indicating this beneficial element via fertigation for the cultivation of forage without P imbalances, especially if cultivated in Oxisol in an irrigated system.

Conclusions and future perspective
Fertigation with Si nanoparticles promotes the absorption of the element by the forage, improves C:N:P homeostasis and the nutritional efficiency of plants cultivated under P deficiency and excess P, and reduces plant senescence regardless of P level, attenuating the stress caused especially by P toxicity in Entisol and in Oxisol, as well as in plants without nutritional stress grown in Oxisol. www.nature.com/scientificreports/ The supply of Si increases the dry mass production of plants in the excessive state of P grown in Entisol, and also in plants grown under adequate and excessive states of P in Oxissol. Leaf senescence showed a lower value with addition of Si in plants grown in Oxisol in the three nutritional states of P.
The research revealed that using Si via fertigation triggers modifications in C:N:P stoichiometry that are relevant to the cultivation of M. maximus cv. Zuri, although the magnitude of the effects depends on the growth cycle, soil, and P nutritional status.
This study opens the way to expand research on the potential of Si via fertigation in forages of other species and also in other soils, having global implications in view of the occurrence of P deficiency and toxicity, which undermine the sustainability of forage cultivation in many countries.

Data availability
Datasets generated or analyzed during the present study are available from the corresponding author upon reasonable request.